Fungal pathogens have a significant and increasing impact on human health-, and the lack of effective treatments highlight the need for further study. This proposal will examine both major pathogens with large clinical impacts and recent cases of severe outbreaks of fungal infections. In the first aim, we will examine two large clinical cohorts of Cryptococcus neoformans, to define the genetic determinants of virulence and niche adaptation in the pathogen. We will also characterize pathogen gene expression at the primary site of severe infection, and both screen for and evaluate mutations that stabilize extra copies of chromosomes, a major mechanism of fungal drug resistance. In the second aim, we will examine three recent cases of fungal outbreaks in the US; isolates of Cryptococcus gattii and Fusarium spp will be sampled from clinical cases and the environment and sequenced to examine how environmental reservoirs differ from strains virulent in humans, similar to work in aim 1, and further monitor how one of these outbreaks spread over geography and time. Lastly in aim 3, we will examine the host and pathogen interface for C. albicans, a common commensal and the most common fungal pathogen, and other commonly observed Candida species infecting the primary innate immune cells involved in early Candida detection and response. We will use network analysis to identify host-pathogen hubs and to study how these networks evolve between strains and species differing in virulence and drug resistance. While these aims represent independent projects to examine fungal pathogenesis, each utilizes large-scale sequencing to extend and develop new paradigms for genomic analysis of fungal virulence.